Project 1 will focus on testing specific hypotheses related to the subcellular localizations of two multiprotem complexes anchored to cell membranes through their binding to PtdIns(3)P and PtdIns(3,4,5)P3, respectively. The PI 3-kinases that produce these 3' phosphoinositides control many signaling and membrane trafficking pathways, but the cellular locations of these kinases with respect to both their substrates and products are virtually completely unknown. Thus, experiments that will be performed in conjunction with Projects 2 and 3 and the Biomedical Imaging Core are designed to localize the CPK and VPS34 PI 3-kinases that produce PtdIns(3)P as well as the p110/p85-type PI 3-kinases that produce PtdIns(3,4,5)P3 using GFP fusion proteins (live and fixed cells) or antibodies (fixed cells) against these kinases. In turn, the subcellular localizations of the PI 3 kinase products PtdIns(3)P and PtdIns (3,4,5,)P3 as well as the substrate PtdIns(4,5)P2 will be defined. This will be pursued using GFP-fusions of FYVE and PH domains that bind PtdIns(3)P and PtdIns(3,4,5)P3 respectively, that our laboratory (Klarlund et al, (l997) Science 275, 1927-1933) and the Corvera laboratory (Patki et al, (1998) Nature 394, 433-434) have recently discovered, and the PLC6PH domain that binds PtdIns(4,5)P2. These experiments will use digital imaging micrcoscopy with enhanced laser illumination and advanced deconvolution algorithms to achieve nanometer resolution. We will thus be able to test the extent to which PtdIns(3)P and PtdIns (3,4,5)P3 co-localize with the P1 3-kinases that produce them, and will use FRET analysis to confirm juxtaposition of these species. Parallel experiments will localize the subcellular disposition of the multiprotein complex containing GRPl (a PH and Sec7 domain protein), its partner GRSPl (an ERM domain protein) and Arf GTPase, which is recruited to PtdIns (3,4,5)P3, and thus compare the localization of this 3'phosphoinositide with its effector complex. We will also visualize in real time, in live cells, the dynamics of movement of this multiprotein complex to sites of cellular PtdIns (3,4,5)P3. Finally, we will collaborate with Project 3 to facilitate the determination of 3D protein structures of the GRPI, Arf GTPase, GRSPI complex that is anchored at membrane sites in association with PtdIns (3,4,5)P3. Taken together, these experiments will provide novel and detailed information about the spatial and structural relationships between 3' phosphoinositides, the enzymes that produce them, and a multiprotein complex bound to membranes via PtdIns (3,4,5)P3 and potentially other membrane embedded components.